CAREER: Discovering Upstream Effectors to Cell Fate Determination

在脊椎动物胚胎的正常发育过程中，胚胎细胞必须发育成生物体的所有不同类型的组织，但有助于组织形成的全套生物分子是未知的。该项目利用分析化学领域最近才开发的先进仪器，确定在早期发育的青蛙胚胎中，单个细胞形成不同类型组织时小分子的变化，这是脊椎动物胚胎发育的重要模型。由此产生的数据将提供以前无法获得的对细胞，组织，器官和生物体形成重要的基本生物过程的见解。这些研究工作作为在主办大学，以及会议，研讨会和国家培训中心，如冷泉港实验室，纽约州的不同参与者的跨学科培训的基础。 这项工作将培养新一代的生物学和化学科学家，包括许多传统上在科学界代表性不足的人群，使他们能够解决当前的挑战，同时也提出这些领域的新问题，以更好地了解正常的脊椎动物胚胎发育和疾病。但小分子（称为代谢物）如何参与细胞发育过程尚不完全清楚。PI的实验室最近发现了能够改变早期青蛙（非洲爪蟾）胚胎中选择干细胞的正常背腹命运的代谢物，表明这些分子也是脊椎动物身体模式化过程中的活跃参与者。这项工作的总体目标是确定代谢物诱导的细胞命运决定的作用机制。这将通过系统细胞生物学方法实现，其中将使用PI实验室开发和验证的独特单细胞质谱技术表征代谢物注射细胞的分子状态。该项目将确定改变细胞命运的代谢物如何扰乱近距离代谢网络以及代谢的关键蛋白质和已知的背腹规格的信号通路。两个单细胞质谱仪器将用于对注射的代谢物进行通量分析，并测量荧光跟踪的细胞克隆中靶蛋白的相对翻译，所述细胞克隆是从活青蛙胚胎中的代谢物注射细胞形成的。所得数据将通过基因敲低实验鉴定用于功能测试的候选基因，以验证代谢物诱导细胞命运变化的拟议作用机制。了解小分子对细胞命运承诺的影响在生命科学的不同领域产生了广泛的影响。 这项工作还将在生物学和分析化学的技术衔接方面对代表性不足的群体进行培训。

Non-Technical ParagraphDuring normal development of the vertebrate embryo, cells of the embryo must develop into all the different types of tissues of the organism, but the complete set of biomolecules contributing to tissue formation is unknown. This project utilizes advanced instruments that have only recently been developed in analytical chemistry to determine changes in small molecules as individual cells form different types of tissues in the early developing frog embryo, which is an important model of vertebrate embryo development. The resulting data will provide previously unavailable insights into basic biological processes important for the formation of cells, tissues, organs, and organisms. These research efforts serve as the foundation for interdisciplinary training of diverse participants at the host university, as well as conferences, seminars, and national training centers such as Cold Spring Harbor Laboratory, NY. This work will train a new generation of scientists in both biology and chemistry, including many traditionally underrepresented populations in science, to allow them to address current challenges but also to ask new questions in these fields to better understand normal vertebrate embryo development and diseases.Technical ParagraphDecades of research has uncovered many genes and gene products with critical roles during development of the vertebrate embryo, but how small molecules (called metabolites) participate in cell developmental processes is not fully known. The PI's laboratory recently discovered metabolites capable of altering the normal dorsal-ventral fate of select stem cells in the early frog (Xenopus laevis) embryo, demonstrating that these molecules, too, are active players during patterning of the vertebrate body. The overall goal of this work is to determine the mechanism of action underlying metabolite-induced cell fate decisions. This will be accomplished through a systems cell biology approach, in which the molecular state of metabolite-injected cells will be characterized using unique single-cell mass spectrometry technologies that were developed and validated in the PI's laboratory. The project will identify how cell-fate altering metabolites perturb close-proximity metabolic networks as well as key proteins of metabolism and known signaling pathways of dorsal-ventral specification. Two single-cell mass spectrometry instruments will be used to perform flux analysis for the injected metabolites and to measure the relative translation of targeted proteins in fluorescently tracked cell clones that form from the metabolite-injected cells in the living frog embryo. The resulting data will identify gene candidates for functional tests via gene knock-down experiments to validate the proposed mechanism of action for metabolite-induced cell fate changes. Understanding small-molecule effects on cell fate commitment raises broad implications in diverse areas of the life sciences. The work will also train underrepresented groups in techniques bridging biology and analytical chemistry.